This invention concerns a method for extracting and purifying genomic DNA from human whole blood a biological cell source, in particular from.
More specifically, the invention provides a method which can be reproduced manually and partly automatically in limited performance times.
The known methods employed for the purification of genomic DNA are complex and time consuming. Routine protocols in current use require at least a crude cell isolation or a nuclei isolation. Moreover, after obtaining the respective "nuclear preparations", most protocols involves long digestions with proteinases, followed by several extractions with phenol. (See for example Molecular Cloning, A Laboratory Manual, Maniatis et al, pg. 9,17-9,18 and Nucl. Acids Res. 3, pg.2303-2306).
Due to the inherent limitations (many careful manipulation of the samples are needed), alternative methods have been developed to optimize the quality and the yield of DNA. These methods are generally based on the use of chaotropic reagents to lyse "nuclear preparations" derived from buffy coats, cells, tissues (See Nucl. Acid Res. 15, 9611, 1987 and Anal. Biochem. 180, 276-278, 1989).
U.S. Pat. No. 5,010,183 discloses purifying DNA and RNA from a biological mixture by simultaneously precipitating them with a cationic detergent.
No references are known to the Applicant disclosing methods that start the preparation from whole blood, thus minimizing the risk of accidental exposure to hazardous viruses during the steps needed to obtain the purified leukocytes or nuclei preparations.
A known "DNA Extractor" apparatus of the Applied Biosystem (Foster City, Calif.), is not satisfactory owing to its low percentage yield and the slowness of the process, based on the chemistry disclosed in Nucleic Acids Res. 3, 2303-2306, 1976: it employs as a reagent phenol, which is expensive, irritant and neurotoxic, and proteinase K, which involves long digestions of the sample and is expensive.
The object of the present applicant is therefore that of providing a method which enables higher percentage yields of pure DNA to be achieved more simply in very short periods of time.